Methods to assess helicase and translocation activities of human nuclear RNA exosome and RNA adaptor complexes

The nuclear RNA exosome collaborates with the MTR4 helicase and RNA adaptor complexes to process, surveil, and degrade RNA. Here we outline methods to characterize RNA translocation and strand displacement by exosome-associated helicases and adaptor complexes using fluorescence-based strand displacement assays. The design and preparation of substrates suitable for analysis of helicase and decay activities of reconstituted MTR4–exosome complexes are described. To aid structural and biophysical studies, we present strategies for engineering substrates that can stall helicases during translocation, providing a means to capture snapshots of interactions and molecular steps involved in substrate translocation and delivery to the exosome.


Introduction
The RNA exosome is a conserved, eukaryotic 3′-to-5′ ribonuclease protein complex that is responsible for several aspects of nuclear and cytoplasmic RNA metabolism, ranging from general RNA turnover to processing and quality control of various coding and noncoding transcripts (Houseley, LaCava, & Tollervey, 2006;Puno, Weick, Das, & Lima, 2019). The human nuclear RNA exosome is composed of 9 core subunits with three S1/KH-domain proteins (EXOSC1-3) atop a hexameric ring of PH-domain proteins (EXOSC4-9) (Fig.  1). The exosome core contains a central channel that guides single-stranded RNA to two RNA-degrading enzymes: DIS3, a processive 3′-to-5′ RNAse II-like exoribonuclease and EXOSC10, a distributive 3′-to-5′ RNase D-like exoribonuclease that associate with the exosome core (Gerlach et al., 2018;Liu, Greimann, & Lima, 2006;Makino, Halbach, & Conti, 2013;Wasmuth, Januszyk, & Lima, 2014;Weick et al., 2018;). This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
MTR4 is a central component of several nuclear RNA adaptor complexes that identify, capture and prime exosome substrates for decay. In human, these include the Nuclear Exosome Targeting (NEXT) complex, the TRF4-2/PAPD5-ZCCHC7-MTR4 polyadenylation (TRAMP) complex, and the Poly(A) Exosome Targeting (PAXT) connection or Polysome Protector complex (PPC) (Fig. 1) (Lubas et al., 2011;Meola et al., 2016;Ogami et al., 2017). NEXT, composed of an RNA binding protein RBM7, a zinc-knuckle protein ZCCHC8, and the MTR4 helicase, functions in turnover of unwanted transcripts produced by pervasive transcription and surveillance of aberrant RNA species that emerge from faulty transcription. PAXT/PPC core is a binary complex between MTR4 and ZFC3H1 and is loosely associated other cofactors that include poly(A) binding protein PABPN1. PAXT/PPC primarily targets processed polyadenylated transcripts. In contrast to its S. cerevisiae counterpart, less is known about endogenous targets of human TRAMP.
The ability of MTR4 and RNA adaptor complexes to disrupt RNA structures and displace proteins deposited on RNA is key to their function in promoting exosome-mediated decay (Puno et al., 2019;Weick & Lima, 2021). In this chapter, we present methods used in our laboratory to characterize helicase activities of exosome-associated helicases and RNA adaptor complexes to determine mechanisms and kinetic models underpinning substrate selection, translocation, unwinding, and targeting to the exosome. These assays may also prove useful in studies aimed at assessing helicases and RNA adaptor complexes for their ability to displace proteins bound to RNA. We also provide a protocol for analyzing helicase-dependent RNA decay by MTR4-exosome complexes. Finally, we describe strategies in designing substrates for trapping helicases during translocation to generate samples for biophysical and structural analysis.

RNA helicase assays with MTR4 and RNA adaptor complexes
Assays that detect strand displacement are widely used to query translocation and/or helicase activity in vitro. These assays measure the ability of a helicase to displace and release an oligonucleotide strand (reporter strand) from a duplex region of a substrate. Unwinding polarity and requirements for a single-stranded translocation strand can be assessed by designing duplex substrates with single-stranded regions extending from either 5′ or 3′ ends.
Several approaches are employed to monitor strand displacement using radiolabeled or fluorescently modified oligonucleotides. In a gel shift assay, the displaced reporter strand is captured by a complementary DNA oligonucleotide to prevent reannealing to the translocation strand while capturing the product for detection using native polyacrylamide gel electrophoresis ( Fig. 2A and B). This is a common and accessible approach, since it utilizes materials and equipment available in most molecular biology laboratories, but it is limited by the number of samples and individual time points that can be conveniently taken manually. Throughput and temporal resolution are enhanced by using Fluorescence Resonance Energy Transfer (FRET)-based assays that enable detection of strand displacement in real time (Özeş, Feoktistova, Avanzino, Baldwin, & Fraser, 2014). Kinetic data can be obtained in minutes instead of hours as is often required for shift assays where assays are followed by gel electrophoresis and scanning. FRET substrates typically consist of a fluorescent reporter strand annealed to a translocation strand conjugated with a quencher dye ( Fig. 2A). Upon strand displacement, the fluorophore is now distant from the quencher resulting in an increase in total reporter fluorescence.
A variant of the FRET-based method, the Molecular Beacon Helicase Assay (MBHA), employs a displacement strand that forms a stem loop after being released from the translocation strand. The displacement strand (molecular beacon) is modified with the fluorophore and quencher moieties at opposite ends (Belon & Frick, 2008;Özeş et al., 2014). Strand displacement results in a decrease in total fluorescence that can be detected in real time ( Fig. 2A and C). The molecular beacon cannot reanneal to the translocation strand, so this approach effectively eliminates the need for a capture strand in the reaction. It also bypasses the synthesis of two modified oligonucleotides, reducing cost and expanding the size range of synthetic translocation strands that can be used.
The assays described in this section are performed under multiple turnover conditions whereby the helicase sequentially binds multiple substrates. For single turnover conditions, a helicase trap oligonucleotide is included in the reactions to prevent rebinding of dissociated helicase to the substrate (Pang, Jankowsky, Planet, & Pyle, 2002). Enzyme titrations are performed with concentrations generally ranging from 10-fold above and below the estimated RNA binding constant and can be used to calculate maximal rate and apparent substrate affinity (half maximal rate concentration).

2.
Incubate tube with resuspended RNA in ThermoMixer at 25 °C with a shake speed of 1000 rpm for 5 min. Transfer resuspended RNA into a fresh microcentrifuge tube.

a.
For the gel shift assay, mix oligonucleotide pairs GS1 and GS2 to generate the 3′ A 20 tailed RNA duplex substrate. Prepare a 10X gel shift unwound control that will be used as a marker for captured reporter strand product: mix 19.5 μl annealing buffer, 0.5 μl of 8 μM GS1 and 20 μl 8 μM Capture1.

b.
For the molecular beacon assay, mix oligonucleotide pairs MBHA1 and MBHA2 to generate the 3′ A 20 tailed RNA duplex substrate. Prepare a 100X molecular beacon control: mix 35 μl annealing buffer and 5 μl of 8 μM MBHA1.

6.
Using Bio-Rad C1000 Touch Thermal Cycler, heat the RNA mixture to 95 °C for 5 min followed by cooling to 16 °C for 10 minutes and 4 °C overnight. Transfer RNA mixture to a microcentrifuge tube and store at −80 °C until needed.

2.
Add 15 μl of 100 nM RNA solution to reaction tube. Mix well.

3.
Prepare a 2X helicase solution in 20 mM Tris pH 7.0, 100 mM NaCl, 0.1 mM TCEP. Add 75 μl of 2X helicase solution in reaction mixture. Mix and incubate for 5 minutes at 22 °C. A protein titration is recommended with concentrations approximately 10-fold below and above the estimated equilibrium binding constant.

4.
For the zero time point, pipet 18 μl reaction mixture into a microcentrifuge tube and add 2 μl 4 μM capture strand in annealing buffer and 20 μl quench solution.

5.
Transfer 90 μl of the reaction mixture to a fresh microcentrifuge tube.

7.
Initiate the reaction by adding 10 μl of 10X ATP-capture solution. Incubate the reaction tube in a ThermoMixer at 30 °C. Optional: Set shake speed to 30 rpm.

8.
Take 18 μl aliquot of reaction mixture per time point and immediately transfer to a tube containing 18 μl 2X quench solution. Mix well.

9.
Incubate quenched samples at 30 °C for 10 minutes to complete proteinase K digestion.

11.
Remove the gel from the cassette and scan for 6-FAM fluorescence (473 nm laser, LPB filter) using Typhoon FLA 9500 scanner. Save the scanned image as tif file.

12.
Open tif file in ImageJ and quantify band intensities of unreacted substrate (I unreacted ) and unwound product (I unwound ). Subtract background fluorescence from each intensities.

14.
Normalize data by subtracting fraction unwound at time = 0 due to nonenzymatic strand displacement from fraction unwound at each time point.

16.
By plotting normalized fraction unwound (f norm ) versus time (t), the data can be fitted to Eq.
(3) (one-phase association equation in GraphPad Prism) to determine the observed unwinding rate constant k obs and reaction amplitude (A):

15.
Initial unwinding rate can be calculated by taking the product of observed rate constant and reaction amplitude or by the taking the slope of fitted linear curve of data at early time points.

2.
Pipet 20 μl MBHA master mix per reaction well into Corning 3693 half-area 96well white plate. Use a multichannel pipette when conducting multiple reactions.

6.
Initiate the reaction by adding 5 μl 20 mM ATP·MgCl 2 solution into each well using a multichannel pipette.

7.
Record fluorescence every 30 s for 40 min at 30 °C.

8.
Calculate normalized fraction unwound for each time point using Eq (4): where S t is the sample fluorescence at time t, S 0 is the sample fluorescence at time 0, M t is the fluorescence of the molecular beacon control at time t, C t is the fluorescence of no helicase control at time t and C 0 is the fluorescence of no protein/helicase control at time 0.

9.
Data are fitted to Eq. (3) using GraphPad Prism to determine the observed unwinding rate constant k obs and the extent of the reaction (A).

10.
Initial rate can be calculated by taking the product of the observed rate constant and reaction amplitude or by the taking the slope of fitted linear curve of data at early time points.

Notes
• Buffer conditions (buffer, pH, ionic strength) should be optimized for every helicase assayed.

•
ATP·MgCl 2 stock solution (200 mM) is prepared using ATP disodium salt hydrate lyophilized powder (Sigma). Hydrate content should be checked in the accompanying analysis certificate. Adjust the pH using potassium hydroxide and supplement with 1 mM Tris-HCl pH 7.0. Add stoichiometric amount of MgCl 2 prior to storage at −80 °C.
• Fluorescence intensity of specific dyes can be reduced when placed adjacent to a guanine nucleotide.

•
The influence of the position (5′ vs 3′ end of the reporter strand) of the fluorophore on helicase activity should be tested.
• Strand displacement assays are typically accompanied with measurements of nucleotide triphosphate (NTP) consumption to determine how energy expenditure is coupled to translocation. For some helicases, NTP hydrolysis is dispensable for strand displacement but is required for enzyme recycling (Liu et al., 2008). The number of NTP used per nucleotide translocation also vary among helicases. Several methods (Brune et al., 1994, Bradley and De La Cruz, 2012, Rajagopal and Lorsch, 2013, Özeş et al., 2014 and commercial kits are available to monitor NTPase activity.

RNA unwinding and decay assays of MTR4-exosome complexes
RNA unwinding by MTR4 and RNA adaptor complexes ultimately leads to 3′ end trimming or decay by the exosome. These processes are coupled through physical interaction of MTR4 with the exosome via nuclear co-factors MPP6 and/or the C1D/EXOSC10 heterodimer. To analyze RNA unwinding and decay activities of recombinant human MTR4exosome complexes, we designed fluorophore-labeled tripartite substrates (Fig. 3, Table  1) that can be used for gel-based assays (Weick et al., 2018). For decay assays, the translocation strand (strand A) is labeled with a fluorescent moiety whereas for helicase assays, a DNA oligonucleotide (strand B) annealed to the translocation strand is conjugated with a fluorophore (Fig. 3). A short poly(A) 8 overhang is included for interaction with MTR4 (Jia et al., 2011). These substrates can serve as templates for exploring conditions or modifications that alter MTR4-exosome activities. The following procedures describe substrate preparation and protocols for helicase and decay assays of MTR4-exosome complexes.

2.
Prepare a stock solution of 32 μM for each oligonucleotide.

4.
Using Bio-Rad C1000 Touch Thermal Cycler, heat the RNA mixture to 95 °C for 5 min followed by cooling to 16 °C for 10 minutes and 4 °C overnight. Transfer RNA mixture to a microcentrifuge tube and store at −80 °C until needed.

3.
For sample at 0 time point, take 18 μl reaction mixture and mix with 2 μl assay buffer and 10 μl quench solution.

4.
Transfer 54 μl reaction mixture to a fresh microcentrifuge tube.

7.
Take 10 μl aliquot of reaction mixture at various time points and terminate the reaction by adding 5 μl quench solution.

8.
Incubate quenched samples at 30 °C overnight to complete proteinase K digestion.

10.
Place the gel cassette in XCell SureLock Mini-Cell tank and fill the upper and lower chambers with 0.5X TBE running buffer (pre-chilled to 4 °C). Flush each well with 0.5X TBE running buffer.

13.
Remove the gel from the cassette and scan for 6-FAM fluorescence (473 nm laser, LPB filter) using Typhoon FLA 9500 scanner.

3.
For sample at 0 time point, take 18 μl aliquot of reaction mixture and mix with 2 μl assay buffer and 10 μl quench solution.

6.
Take 10 μl aliquot of reaction mixture at various time points and stop the reaction by adding 5 μl quench solution.

7.
Incubate quenched samples at 30 °C overnight to complete proteinase K digestion.

9.
Place the gel cassette in XCell SureLock Mini-Cell tank and fill the upper and lower chambers with 0.5X TBE running buffer. Flush each well with 0.5X TBE running buffer.

12.
Remove the gel from the cassette and scan for fluorescein fluorescence (473 nm laser, LPB filter)using Typhoon FLA 9500 scanner.

Preparation of RNA-loaded human MTR4-exosome complex for structural studies
To understand how MTR4 and other exosome-associated helicases unwind and feed RNA into the exosome, we engineered substrates to capture the helicase during the course of RNA translocation. We previously reported use of a substrate with a chimeric DNA-RNA translocation strand (Fig. 4a) to determine the cryogenic electron microscopy (cryo-EM) structure of RNA-loaded human MTR4-exosome complex in the process of unwinding and delivering RNA to the exosome (Weick et al., 2018). The DNA-RNA translocation strand allows MTR4 tracking on the RNA segment of the substrate but induces stalling as the helicase reaches the DNA-RNA junction (Fig. 4b). We designed the length of the translocation strand based on the number of nucleotides accommodated by MTR4, exosome core, and DIS3 in reported crystal structures of S. cerevisiae homologs (Weir et al., 2010, Zinder et al., 2016. The optimal length was refined by performing a helicase reaction followed by a crosslinking assay (Weick et al., 2018). In this section, we describe the preparation and loading of a tripartite DNA-RNA chimera substate to MTR4-exosome complex for cryo-EM analysis.

9.
Take peak fractions and supplement with 2 mM AMPPNP using quenching buffer. RNA loaded human MTR4-exosome complex can be used for subsequent cryo-EM grid preparation and analysis.

Notes
• Bulky chemical moieties can be conjugated to a nucleotide in the translocation strand to block MTR4 tracking on the RNA substrate. For example, we found that incorporation of 2′-amino-butyryl-pyrene-modified uridine (2′pyU) in the translocation strand inhibits unwinding by NEXT (Fig. 5). Human RNA exosome subunits, co-factors, and RNA adaptor complexes.   Substrate design and reaction scheme for RNA helicase and decay assay of human MTR4exosome complex.